Compositions and methods for sensitizing and inhibiting growth of human tumor cells

ABSTRACT

Polynucleotides encoding carboxylesterase enzymes and polypeptides encoded by the polynucleotides which are capable of metabolizing a chemotherapeutic prodrug and inactive metabolites thereof to active drug are provided. Compositions and methods for sensitizing tumor cells to a prodrug chemotherapeutic agent and inhibiting tumor growth with this enzyme are also provided. In addition, screening assay for identification of drugs activated by this enzyme are described.

This application is a continuation-in-part of PCT/US99/03171 filed Feb.12, 1999, which claims the benefit of priority from provisional U.S.Application Serial No. 60/075,258, filed Feb. 19, 1998.

This invention was supported in part by funds from the U.S. GovernmentNIH Grant Nos. CA-66124 and CA-63512 and the U.S. Government maytherefore have certain rights in the invention.

INTRODUCTION

1. Field of the Invention

This invention relates to novel polynucleotides identified and sequencedwhich encode a carboxylesterase enzyme, polypeptides encoded by thesepolynucleotides and vectors and host cells comprising these vectorswhich express the enzyme. This enzyme is capable of metabolizingchemotherapeutic prodrugs and inactive metabolites into active drug. Theinstant invention thus relates to compositions comprising thesepolynucleotides and methods for sensitizing selected tumor cells to achemotherapeutic prodrug by transfecting the tumor cells with apolynucleotide placed under the control of a disease-specific responsivepromoter. Sensitized tumor cells can then be contacted with achemotherapeutic prodrug to inhibit tumor cell growth. Compositions ofthe present invention can also be used in combination withchemotherapeutic prodrugs to purge bone marrow of tumor cells. Theinvention further includes novel drug screening assays for identifyingchemotherapeutic prodrugs that are activated by this enzyme.

2. Background of the Invention

Cancer is a disease resulting from multiple changes at the genomiclevel. These changes ultimately lead to the malfunction of cell cyclemachinery and finally to autonomous cell proliferation. Neoplastictransformation involves four types of genes: oncogenes, tumor-suppressorgenes, mutator genes, and apoptotic genes. Different types of cancer caninvolve alteration of any one or any combination of these genes.

Proto-oncogenes of the myc family are overexpressed in many differenttypes of human tumors including tumors of the breast, colon, cervix,head and neck, and brain. Many solid tumors amplify or overexpressc-myc, with up to a 50-fold increase in c-myc RNA in tumor cellsrelative to normal cells having been reported (Yamada, H. et al. 1986.Jpn. J. Cancer Res. 77:370-375). For example, three of the six mostcommon solid tumors, including up to 100% of colon adenocarcinomas, 57%of breast cancers, and 35% of cervical cancers, demonstrate increasedlevels of c-myc protein. Enforced expression of c-myc in nontumorigeniccells causes immortalization but not transformation; however, elevatedlevels of c-myc protein are rare in benign cancers and normaldifferentiated tissue. While solid tumors can oftentimes be removedsurgically, overexpression of c-myc has been linked with amplificationof the c-myc gene and correlated with poor prognosis and an increasedrisk of relapse (Nagai, M. A. et al. 1992. Dis. Colon Rectum 35:444-451;Orian, J. M. et al. 1992. Br. J. Cancer 66:106-112; Riou, G. et al.1987. Lancet 2:761-763; Field, J. K. et al. 1989. Oncogene 4:1463-1468).

Another member of the myc oncogene family, N-myc, has been linked withdevelopment of neuroblastomas in young children. Overexpression of thismember of the myc family of proto-oncogenes has also been correlatedwith advanced stages of disease and poor prognosis (Brodeur, G. M. etal. 1997. J. Ped. Hematol. Oncol. 19:93-101). Primary tumors for thisspecific condition usually arise in the abdomen and as many as 70% ofpatients have bone marrow metastases at diagnosis (Matthay, K. E. 1997.Oncology 11:1857-1875). Treatment of children with Stage 4 disease usingsurgery, chemotherapy, and purged autologous or allogeneic marrowtransplant produces a progression-free survival rate of 25 to 49% inpatients four years post transplant (Matthay, K. K. et al. 1994. J.Clin. Oncol. 12:2382-2389). Most relapses after autotransplant occur atsites of bulk disease and/or previously involved sites. Estimates of therate of local recurrence vary depending upon the study. However,recurrence of tumor at an original site has been estimated to occur inapproximately 25% of high risk neuroblastoma patients.

Further, definitive evidence from gene marking studies indicates thatautologous marrow, free of malignant cells by standard clinical andmorphologic criteria, contributes to relapse at both medullary andextramedullary sites (Rill, D. R. et al. 1994. Blood 84:380-383). In arecent pilot clinical study, bone marrow involvement at diagnosiscorrelated with specific relapse at that site in children receivingautologous purged marrow (Matthay, K. K. et al. 1993. J. Clin. Oncol.11:2226-2233). Accordingly, improvements in surgery, detection of tumormargins, development of new anticancer drugs or application of noveltherapies are required to prevent local tumor regrowth. In particular,more effective treatment strategies are needed for elimination of“minimal residual disease” or “MRD” which results from the presence of asmall number of tumor cells at the site of disease after treatments suchas tumor resection or purging bone marrow of tumor cells.

CPT-11 (irinotecan,7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) is aprodrug currently under investigation for the treatment of cancer thatis converted to the active drug known as SN-38(7-ethyl-10-hydroxy-camptothecin) (Tsuji, T. et al. 1991. J.Pharmacobiol. Dynamics 14:341-349; Satoh, T. et al. 1994. Biol. Pharm.Bull. 17:662-664). SN-38 is a potent inhibitor of topoisomerase I(Tanizawa, A. et al. 1994. J. Natl. Cancer Inst. 86:836-842; Kawato, Y.et al. 1991. Cancer Res. 51:4187-4194), an enzyme whose inhibition incells can result in DNA damage and induction of apoptosis (Hsiang, Y.-H. et al. 1989. Cancer Res. 49:5077-5082). The specific enzymeresponsible for activation in vivo of CPT-11 has not been identified,although serum or liver homogenates from several mammalian species havebeen shown to contain activities that convert CPT-11 to SN-38 (Tsuji, T.et al. 1991. J. Pharmacobiol. Dynamics 14:341-349; Senter, P. D. et al.1996. Cancer Res. 56:1471-1474; Satoh, T. et al. 1994. Biol. Pharm.Bull. 17:662-664). Uniformly, these activities have characteristics ofcarboxylesterase (CE) enzymes (Tsuji, T. et al. 1991. J. Pharmacobiol.Dynamics 14:341-349; Senter, P. D. et al. 1996. Cancer Res.56:1471-1474; Satoh, T. et al. 1994. Biol. Pharm. Bull. 17:662-664). Infact, SN-38 can be detected in the plasma of animals and humans minutesafter the administration of CPT-11 (Stewart, C. F. et al. 1997. CancerChemother. Pharmacol. 40:259-265; Kaneda, N. et al. 1990. Cancer Res.50:1715-1720; Rowinsky, E. K. et al. 1994. Cancer Res. 54:427-436),suggesting that a CE enzyme present in either serum or tissues canconvert the camptothecin analog to its active metabolite.

CEs are ubiquitous serine esterase enzymes that are thought to beinvolved in the detoxification of a variety of xenobiotics. CEs areprimarily present in liver and serum, however, the physiological role ofthis class of enzymes has yet to be identified. A recent biochemicalanalysis of 13 CEs compared their ability to metabolize CPT-11 to SN-38.While the efficiency of conversion varied between enzymes, thoseisolated from rodents were the most efficient (Satoh, T. et al. 1994.Biol. Pharm. Bull. 17:662-664). The amino acid sequence of a rabbitliver CE has been disclosed (Korza, G. and J. Ozols. 1988. J. Biol.Chem. 263:3486-3495). In addition, there are currently 13 CDNA sequencesencoding CE in the GenBank and EMBL databases, including a rat serum andrat liver microsomal CE. Interestingly, CEs purified from human tissuesdemonstrated the least efficient conversion of CPT-11 to SN-38, withless than 5% of the prodrug being converted to active drug (Leinweber,F. J. 1987. Drug Metab. Rev. 18:379-439; Rivory, L. P. et al. 1997.Clin. Cancer Res. 3:1261-1266).

In addition to metabolism to SN-38, in humans CPT-11 is also metabolizedto a compound known as APC (Haaz, M. C. et al. 1998. Cancer Res.58:468-472). APC has little, if any, anti-tumor activity and is notconverted to an active metabolite in humans (Rivory, L. P. et al. 1996.Cancer Res. 56:3689-3694).

In preclinical studies, CPT-11 administered to immune-deprived micebearing human tumor xenografts produces complete regression ofglioblastomas, rhabdomyosarcomas (RMS), neuroblastomas, and colonadenocarcinomas (Houghton, P. J. et al. 1995. Cancer Chemother.Pharmacol. 36:393-403; Houghton, P. J. et al. 1993. Cancer Res.53:2823-2829). However, maintenance of tumor regression in studies withCPT-11 appears to be dependent upon drug scheduling, suggesting thatviable tumor cells survive therapy (i.e., minimal residual disease(MRD)). These studies also showed a steep dose-response relationshipbetween dose of drug administered and induction of tumor regression. Forexample, 20 mg of CPT-11/kg/day given daily for 5 days for two weeksproduced complete regression of Rh18 RMS xenografts, while 10 mg/kg/daygiven on the same schedule produced only partial tumor regression.Similar effects were seen when mice bearing SJGC3A colon adenocarcinomaxenografts were treated with 40 mg CPT-11/kg compared to a 20 mg/kgdose.

Early clinical trials with CPT-11 indicate that the prodrug also hasanti-tumor activity in vivo against many different types of solid tumorsin humans. However, myelosuppression and secretory diarrhea limit theamount of drug that can be administered to patients. Accordingly, beforethis promising anti-cancer agent can be used successfully, thesedose-limiting toxicities must be overcome.

The development of new effective treatment strategies for cancer isdependent upon the availability of specific drug screening assays.Specific drug screening assays can involve isolated target tissuemodels, i.e., isolated heart, ileum, vasculature, or liver from animalssuch as rabbits, rats, and guinea pigs, wherein the target tissue isremoved from the animal and a selected activity of that target tissue ismeasured both before and after exposure to the candidate drug. Anexample of a selected activity measured in drug screening assays toidentify new cancer agents is the activity of enzymes such astopoisomerase I or II, which are known to modulate cell death. Suchassays can also be used to screen for potential prodrugs which areconverted to the active metabolite in selected tissues or to identifyselected tissues capable of converting prodrug to its active metabolite.

However, any molecular event that is shown to be modified by a novelclass of compounds can be developed as a screening assay for selectionof the most promising compounds for therapeutic development. In fact, inrecent years the idea of modulating cells at the genomic level has beenapplied to the treatment of diseases such as cancer. Gene therapy fortreatment of cancer has been the focus of multiple clinical trialsapproved by the National Institutes of Health Recombinant DNA AdvisoryCommittee, many of which have demonstrated successful clinicalapplication (Hanania et al. 1995. Am. Jour. Med. 99:537-552; Johnson etal. 1995. J. Am. Acad. Derm. 32(5):689-707; Barnes et al. 1997.Obstetrics and Gynecology 89:145-155; Davis et al. 1996. Current Opinionin Oncology 8:499-508; Roth and Cristiano 1997. J. Natl. Canc. Inst.89(1):21-39). To specifically target malignant cells and spare normaltissue, cancer gene therapies must combine selective gene delivery withspecific gene expression, specific gene product activity, and, possibly,specific drug activation. Significant progress has been made in recentyears using both viral (retrovirus, adenovirus, adeno-associated virus)and nonviral (liposomes, gene gun, injection) methods to efficientlydeliver DNA to tumor sites. Genes can be transfected into cells byphysical means such as scrape loading or ballistic penetration, bychemical means such as coprecipitation of DNA with calcium phosphate orliposomal encapsulation; or by electro-physiological means such aselectroporation. The most widely used methods, however, involvetransduction of genes by means of recombinant viruses, taking advantageof the relative efficiency of viral infection processes. Current methodsof gene therapy involve infection of organisms withreplication-deficient recombinant viruses containing the desired gene.The replication-deficient viruses most commonly used includeretroviruses, adenoviruses, adeno-associated viruses, lentiviruses andherpes viruses. The efficacy of viral-mediated gene transfer canapproach 100%, enabling the potential use of these viruses for thetransduction of cells in vivo.

Adenovirus vector systems in particular have several advantages. Theseinclude the fact that non-dividing cells can be transduced; transducedDNA does not integrate into host cell DNA, thereby negating insertionalmutagenesis; the design of adenoviral vectors allows up to 7 kb offoreign DNA to be incorporated into the viral genome; very high viraltiters can be achieved and stored without loss of infectivity; andappropriate plasmids and packaging cell lines are available for therapid generation of infectious, replication-deficient virus (Yang, N. S.1992. Crit. Rev. Biotechnol. 12:335-356). The effectiveness ofadenoviral-mediated delivery of genes into mammalian cells in cultureand in animals has been demonstrated.

To increase the specificity and safety of gene therapy for treatment ofcancer, expression of the therapeutic gene within the target tissue mustalso be tightly controlled. For tumor treatment, targeted geneexpression has been analyzed using tissue-specific promoters such asbreast, prostate and melanoma specific promoters and disease-specificresponsive promoters such as carcinoembryonic antigen, HER-2/neu,Myc-Max response elements, DF3/MUC. Dachs, D. U. et al. 1997. Oncol.Res. 9(6-7):313-25. For example, the utility of herpes simplex virusthymidine kinase (HSV-TK) gene ligated with four repeats of the Myc-Maxresponse element, CACGTG (SEQ ID NO:22), as a gene therapy agent fortreatment of lung cancer with ganciclovir was examined in c-, L- orN-myc-overexpressing small cell lung cancer (SCLC) cell lines (Kumagai,T. et al. 1996. Cancer Res. 56(2):354-358). Transduction of the HSV-TKgene ligated to this CACGTG (SEQ ID NO:22) core rendered individualclones of all three SCLC lines more sensitive to ganciclovir thanparental cells in vitro, thus suggesting that a CACGTG-driven HSV-TKgene may be useful for the treatment of SCLC overexpressing any type ofmyc family oncogene. Additional experiments with c-myc have focused onthe use of the ornithine decarboxylase (ODC) promoter gene. Within thefirst intron of the ODC gene are two CACGTG “E boxes” that providebinding sites for the c-myc protein when bound to its partner proteinknown as max. Mutation of the E box sequence results in the inability ofc-myc to transactivate the ODC promoter. Previous reports indicate thatreporter constructs containing the ODC promoter fused upstream of thechloramphenicol acetyltransferase gene immediately adjacent to thesecond exon were activated in cells that overexpress c-myc(Bello-Fernandez, C. et al. 1993. Proc. Natl Acad. Sci. USA90:7804-7808). In contrast, transient transfection of promoterconstructs in which the E boxes were mutated (CACGTG (SEQ ID NO:22) toCACCTG (SEQ ID NO:25) demonstrate significantly lower reporter geneactivity. These data suggest that it is possible to activatetranscription of specific genes under control of the c-myc responsiveODC promoter. In the case of N-myc, N-myc protein is a basichelix-loop-helix (BHLH) protein that can dimerize with proteins of thesame class. N-myc dimerizes with the BHLH protein max to form a complexthat binds to the CACGTG motif present in gene promoters, such as ODC,resulting in transactivation and expression of specific genes containingthis sequence (Lutz, W. et al. 1996. Oncogene 13:803-812). Studies in aneuroblastoma cell line and tumors have shown that binding of N-myc toits consensus DNA binding sequence correlates with N-myc expression,data that indicate that the level of N-myc in neuroblastoma cells is adetermining factor in expression of proteins under control of promoterscontaining the CACGTG sequence (Raschella, G. et al. 1994. Cancer Res.54:2251-2255). Inhibition of expression of the c-myc gene via antisenseoligonucleotides as a means for inhibiting tumor growth has also beendisclosed (Kawasaki, H. et al. 1996. Artif. Organs 20(8):836-48).

In the present invention, polynucleotides encoding carboxylesteraseenzymes or active fragments thereof and polypeptides encoded therebywhich are capable of metabolizing the chemotherapeutic prodrug CPT-11and its inactive metabolite APC to active drug SN-38 are disclosed. Useof these enzymes in combination with APC renders this inactivemetabolite a useful chemotherapeutic prodrug. It has also been foundthat compositions comprising a polynucleotide of the present inventionand a disease-specific responsive promoter can be delivered to selectedtumor cells to sensitize the tumor cells to the chemotherapeutic prodrugCPT-11, thereby inhibiting tumor cell growth.

SUMMARY OF THE INVENTION

An object of the present invention is to provide polynucleotidesencoding carboxylesterases capable of metabolizing a chemotherapeuticprodrug and inactive metabolites thereof to active drug.

Another object of the present invention it to provide polypeptidesencoded by these polynucleotides.

Another object of the present invention is to provide vectors comprisingthese polynucleotides and host cells containing these vectors whichexpress carboxylesterases.

Another object of the present invention is to provide a compositioncomprising a polynucleotide encoding a carboxylesterase and adisease-specific responsive promoter of selected tumor cells or apromoter such as CMV.

Another object of the present invention is to provide a method forsensitizing tumor cells to a chemotherapeutic prodrug which comprisestransfecting selected tumor cells with a composition comprising apolynucleotide encoding carboxylesterase and a disease-specificresponsive promoter of the selected tumor cells.

Another object of the present invention is to provide a method ofinhibiting growth of selected tumor cells which comprises sensitizingselected tumor cells to a chemotherapeutic prodrug metabolized to activedrug by a carboxylesterase and administering a chemotherapeutic prodrug.

Another object of the present invention is to provide a method of usingAPC as a prodrug in the treatment of cancer.

Another object of the present invention is to provide drug screeningassays for identification of compounds activated by carboxylesterases.

Yet another object of the present invention is to provide a modifiedornithine decarboxylase promoter which upregulates target proteinexpression in tumor cells that over-express myc proteins.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the alignment of the amino acid sequences of a rabbit livercarboxylesterase (Rab; GenBank Accession # AF036930), a human livercarboxylesterase (hCE1; GenBank Accession # M73499) and the humanintestinal carboxylesterase (hiCE; GenBank Accession # Y09616). Theactive site triad (Ser-240, Glu-364 and His-478) are indicated by anasterisk (*). Identical residues are indicated by a vertical line (|),conservative changes by a colon (:), semi-conservative changes by aperiod (.), and computer inserted gaps within the amino acids areindicated by a dash (-). Large areas of homology between all threeproteins are shaded.

FIG. 2 shows the design of the oligonucleotides used for degenerate PCR.The amino acid sequence (SEQ ID NO:6) and the coding sequence (SEQ IDNO:7) of residues 1 through 5 of rabbit CE are depicted along with thecorresponding oligonucleotide Rab51 (SEQ ID NO:8) and Rab52 (SEQ IDNO:9). Also depicted are the amino acid sequence (SEQ ID NO:10), thecoding sequence (SEQ ID NO:11) and the reverse complement (SEQ ID NO:12)of residues 518 through 524 of rabbit CE, along with oligonucleotideRab31 (SEQ ID NO:13) and Rab32 (SEQ ID NO:14).

FIG. 3 shows the alignment of N-terminal signal sequences of the rabbitliver CE (SEQ ID NO:15) and other known CEs including rat (P10959; SEQID NO:16), human (P23141; SEQ ID NO:17), rat (16303; SEQ ID NO:18) andmouse (P23953;

SEQ ID NO:19). Residues common to all CEs are underlined and the 18residue leader sequence is indicated in italics. The Swissprot Accessionnumbers are indicated in parentheses.

FIG. 4 shows the complete coding sequence of the rabbit liver CE (SEQ IDNO:20) and the amino acid sequence encoded thereby (SEQ ID NO:21). The1698 bp ORF encodes a 62.3 kDa protein. The N-terminal hydrophobicleader sequence is in italics, the 5′ and 3′ RACE sequences areunderlined and the potential active site serine is indicated by anasterisk. The carboxylesterase B-1 and B-2 motifs, at amino acids208-223 and 114-124 are double underlined. Numbers over the sequencerefer to nucleotide position whereas numbers along the left margin referto amino acid residues.

FIG. 5 is a linegraph comparing % cell survival, depicted on the Y-axis,at various concentrations of CPT-11, depicted on the X-axis. ControlCos7 cells (filled squares) are approximately 350-fold more sensitive toCPT-11 than Cos7 cell transfected with CE (filled triangles).

FIG. 6 is a linegraph showing the conversion of APC, depicted on theX-axis at nanomolar concentrations, to SN-38, depicted on the Y-axis atnanomolar concentrations, in vitro by the activity of rabbit liver CEgiven at doses of 0 (filled cross), 10 (filled hexagon), 25 (filledtriangle), 50 (filled circle) or 100 (filled square) units. Datapresented represent the mean response at each dose level.

FIG. 7 is a linegraph showing a comparison of the sensitization,depicted as t survival on the Y-axis, of U-373 glioma cells exposed toAPC, depicted as log [APC] at concentrations from 10⁻⁸ to 10⁻⁵ M on theX-axis, from in situ expression of rabbit liver CE (filled squares) andhuman alveolar macrophage CE (filled circles). Cells were exposed for 2hours to APC.

FIG. 8 provides the chemical structures of CPT-11, APC and SN-38.

FIGS. 9A, 9B, and 9C are linegraphs showing the responses of micebearing Rh30 and Rh30pIRES_(rabbit) rhabdosarcoma xenografts to CPT-11treatment. Each line on each graph shows the growth of an individualtumor. The tumor growth rate is depicted on the Y-axis of each graph interms of tumor volume and is plotted as a function of time in weeks(X-axis). FIG. 9A depicts cells expressing rabbit CE(Rh30pIRES_(rabbit)) not treated with CPT-11. FIG. 9B depicts cellsexpressing rabbit CE (Rh30pIRES_(rabbit)) and then treated with CPT-11and shows complete tumor regression, even out to 12 weeks. FIG. 9Cdepicts control cells (Rh30) exposed to CPT-11 and shows initialregression but regrowth.

FIG. 10 is a linegraph showing the effects of CPT-11 treatment on U373glioblastoma xenografts expressing rabbit CE. Mice bearing xenograftswere treated with CPT-11 (7.5 mg/kg for 5 days) for three treatmentcycles. The tumor growth rate is depicted on the Y-axis in terms oftumor volume and is plotted as a function of time in weeks (X-axis).Open circles depict the tumor volume of untreated U373 xenograftsexpressing rabbit CE. Filled triangles depict the response of controlxenografts (no rabbit CE) treated with CPT-11. Filled squares depict theresponse of cells expressing rabbit CE and treated with CPT-11. The datashow that tumor regression was seen only in treated cells expressingrabbit CE. Each point represents the mean of 14 tumors in 7 individualmice.

FIG. 11 depicts the modifications of the myc-responsive ornithinedecarboxylase promoter where ODC is the ornithine decarboxylasepromoter, R4 and R6 are 4 repeats and 6 repeats, respectively, of themyc-responsive CACGTG E-box sequence, ΔR6 and ΔODC are constructsanalogous to R6 and ODC, respectively, except the E-box sequence hasbeen changed to CACCTG, and CAT is the chloramphenicol acetyltransferasegene.

DETAILED DESCRIPTION OF THE INVENTION

CPT-11 is a promising anti-cancer prodrug, that when given to patients,is converted to its active metabolite SN-38 by a human carboxylesterase.However, conversion in patients is relatively inefficient and less than5% of the prodrug is metabolized to SN-38 (Rivory, L. P. et al. 1997.Clin. Cancer Res. 3:1261-1266). In patients, this prodrug is alsometabolized to APC (Haaz, M-C. et al. 1998. Cancer Res. 58:468-472). APChas little, if any, active anti-tumor activity and is not converted toan active metabolite in humans (Rivory, L. P. et al. 1996. Cancer Res.56:3689-3694). Accordingly, high concentrations of this prodrug must beadministered to achieve effective levels of active drug in vivo.However, myelosuppression and secretory diarrhea limit the amount ofprodrug that can be administered to patients.

In the present invention, a method of sensitizing tumor cells to reducethe effective dose of a prodrug required to inhibit tumor cell growth isprovided which comprises transfecting selected tumor cells with apolynucleotide under the control of a disease-specific responsivepromoter such as a myc promoter. The present invention exploits thetumor-specific overexpression of oncogenes of the myc family to produceselective killing with a chemotherapeutic prodrug.

In accordance with one aspect of the present invention there areprovided polynucleotides which encode carboxylesterases capable ofmetabolizing a chemotherapeutic prodrug and inactive metabolites thereofto active drug. By “polynucleotides” it is meant to include any form ofDNA or RNA such as cDNA or genomic DNA or MRNA, respectively, encodingthese enzymes or an active fragment thereof which are obtained bycloning or produced synthetically by well known chemical techniques. DNAmay be double- or single-stranded. Single-stranded DNA may comprise thecoding or sense strand or the non-coding or antisense strand. Thus, theterm polynucleotide also includes polynucleotides which hybridize understringent conditions to the above-described polynucleotides. As usedherein, the term “stringent conditions” means at least 60% homology athybridization conditions of 60° C. at 2×SSC buffer. In one embodiment,the polynucleotide comprises the cDNA depicted in FIG. 4 (SEQ ID NO:20)or a homologous sequence or fragment thereof which encodes a polypeptidehaving similar activity to that of this rabbit liver CE enzyme. Inanother embodiment, the polynucleotide comprises a cDNA as depicted inSEQ ID NO:27 encoding human intestinal carboxylase as depicted in SEQ IDNO:28. Due to the degeneracy of the genetic code, polynucleotides of thepresent invention may also comprise other nucleic acid sequencesencoding these enzymes and derivatives, variants or active fragmentsthereof. The present invention also relates to variants of thesepolynucleotides which may be naturally occurring, i.e., allelicvariants, or mutants prepared by well known mutagenesis techniques.

Also provided in the present invention are vectors comprisingpolynucleotides of the present invention and host cells which aregenetically engineered with vectors of the present invention to produceCE or active fragments of this enzyme. Generally, any vector suitable tomaintain, propagate or express polynucleotides to produce the enzyme inthe host cell may be used for expression in this regard. In accordancewith this aspect of the invention the vector may be, for example, aplasmid vector, a single- or double-stranded phage vector, or a single-or double-stranded RNA or DNA viral vector. Such vectors include, butare not limited to, chromosomal, episomal and virus-derived vectorse.g., vectors derived from bacterial plasmids, bacteriophages, yeastepisomes, yeast chromosomal elements, and viruses such as baculoviruses,papova viruses, SV40, vaccinia viruses, adenoviruses, fowl pox viruses,pseudorabies viruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, cosmids and phagemids. Selection of anappropriate promoter to direct mRNA transcription and construction ofexpression vectors are well known. In general, however, expressionconstructs will contain sites for transcription initiation andtermination, and, in the transcribed region, a ribosome binding site fortranslation. The coding portion of the mature transcripts expressed bythe constructs will include a translation initiating codon at thebeginning and a termination codon appropriately positioned at the end ofthe polypeptide to be translated. Examples of eukaryotic promotersroutinely used in expression vectors include, but are not limited to,the CMV immediate early promoter, the HSV thymidine kinase promoter, theearly and late SV40 promoters, the promoters of retroviral LTRs, such asthose of the Rous Sarcoma Virus(RSV), and metallothionein promoters,such as the mouse metallothionein-I promoter. Vectors comprising thepolynucleotides can be introduced into host cells using any number ofwell known techniques including infection, transduction, transfection,transvection and transformation. The polynucleotides may be introducedinto a host alone or with additional polynucleotides encoding, forexample, a selectable marker. Host cells for the various expressionconstructs are well known, and those of skill can routinely select ahost cell for expressing the rabbit liver CE enzyme or the humanintestinal CE enzyme in accordance with this aspect of the presentinvention. Examples of mammalian expression systems useful in thepresent invention include, but are not limited to, the C127, 3T3, CHO,HeLa, human kidney 293 and BHK cell lines, and the COS-7 line of monkeykidney fibroblasts. Alternatively, as exemplified herein, rabbit CE canbe expressed in Spodoptera frugiperda Sf21 cells via a baculovirusvector (see Example 3).

The present invention also relates to compositions comprising apolynucleotide of the present invention which have been found to beuseful in sensitizing tumor cells to CPT-11 cytotoxicity by combinationtherapy of the prodrug and a CE enzyme. The present invention thusprovides methods for sensitizing tumor cells to a prodrug oncologicagent. In this context, by “sensitizing” it is meant that the effectivedose of the prodrug can be reduced when the compositions and methods ofthe present invention are employed. In a case where the prodrug'stherapeutic activity is limited by the occurrence of significanttoxicities, or dose-limiting toxicities, sensitization of tumor cells tothe prodrug is especially useful.

In one embodiment, selected tumor cells are transfected with the cDNA ofthe present invention and expressed via a well known promoter such asthe CMV promoter or, more preferably, via a disease-specific responsivepromoter which specifically targets the selected tumor cells. Targetedgene expression in tumor cells has been achieved using disease-specificresponsive promoters such as carcinoembryonic antigen, HER-2/neu,Myc-Max response elements, and DF3/MUC. Thus, a composition comprisingthe cDNA rabbit liver CE or human intestinal CE and a disease-specificresponsive promoter such as these can be used to transfect and sensitizetumor cells containing the disease-specific responsive promoter.Accordingly, the present invention provides a means for exploitingtumor-specific expression associated with a disease-specific responsivepromoter to provide for selective therapy of tumors.

Since myc expression is deregulated in a wide variety of human tumors,myc is an attractive target for chemotherapeutics. No known drugspecifically interacts with either the c-myc or N-myc protein. However,cells overexpressing a myc oncogene can be targeted with compositions ofthe present invention comprising a polynucleotide of the presentinvention under the control of a myc specific promoter. Thus, using thepresent invention the tumor-specific overexpression of c-myc and N-myccan be exploited to produce selective killing with a chemotherapeuticagent. Specifically, transcription of genes under the control of thepromoter containing the CACGTG (SEQ ID NO:22) binding sequence of eitherN-myc or c-myc are upregulated in cells overexpressing these myc genes,producing tumor cell-specific expression of the polynucleotide encodingthe CE that is capable of activating the chemotherapeutic prodrugCPT-11.

The ability of a promoter to regulate gene expression was confirmed incell lines overexpressing c-myc, SJ-G2 and NCI-H82 cells (whichoverexpress c-myc) and Rh28 cells (which have no detectable levels ofc-myc protein). In these experiments, cells were transiently transfectedwith a plasmid containing the ODC promoter controlling expression of areporter gene for chloramphenicol acetyltransferase. A mutated ODCpromoter in which c-myc transactivation domains have been inactivated bypoint mutations was used as a control. A 4- to 5-fold increase inreporter activity was observed in SJ-G2 cells and NCI-H82 cells,respectively, following transfection with the plasmid containing nativeODC promoter as compared to the mutant promoter sequence. No significantincrease in promoter activity was observed in Rh28 cells. These resultsare consistent with c-myc-mediated activation of transcription bybinding to the cognate sequence within the ODC promoter. In addition,the levels of activation were similar to that seen with reporterconstructs when enforced co-expression of c-myc occurs duringtransfection of CV-1 and NIH-3T3 cells.

Additional experiments were conducted to identify disease-specificresponsive promoters for c-myc and n-myc expressing cell lines withoptimal activity. The strategy used in these experiments was to combinea myc-specific promoter with a myc-responsive enhancer. A similarcombination approach with a PSA promoter and enhancer was shown toresult in production of a strong, specific promoter/enhancer forprostate cancer cells (Pang, S. et al. 1995. Human Gene Therapy6:1417-1426; Pang, S. et al. 1997. Cancer res. 57:495-499).Specifically, modified ornithine decarboxylase (ODC) promoters wereconstructed (FIG. 11). The endogenous ODC promoter contains two CACGTGE-box sequences. The modified ODC promoters of the present inventionwere constructed by inclusion of additional CACGTG E-box sequences asfollows: the promoter referred to herein as R4ODC comprised 4 additionalCACGTG E-box sequences 5′ to the endogenous promoter; the promoterreferred to herein as ODCR4 comprised 4 additional CACGTG E-boxsequences 3′ to the endogenous promoter; the promoter referred to hereinas R4ODCR4 comprised 4 additional CACGTG E-box sequences 5′ as well as3′ to the ODC promoter; and the promoter referred to herein R6ODCcomprised 6 additional CACGTG E-box sequences 5′ to the promoter.Accordingly, the constructs contained a total of 6, 6, 10 and 8 CACGTGsites, respectively. Mutated ODC promoters were constructed that encodeda mutated ODC promoter (ΔODC) with a mutated E-box sequence of CACCTG(SEQ ID NO:25). The promoter referred to herein as LR6ODC had 6additional mutated E-box sequences 5′ to ΔODC. The negative controlΔR6ΔODC promoter construct contained a total of 8 modified E-boxsequences. All constructs further comprised the chloramphenicolacetyltransferase (CAT) gene.

The relative transcriptional efficiency of the promoter/enhancerconstructs was evaluated in the c-myc expressing glioblastoma cell lineSJ-G2. The negative control in these experiments was the SJ-G3 cellline. Immunoblots showed that the 64 kDa form of c-myc protein wasreadily detectable in SJ-G2 glioblastoma cells, but not in SJ-G3glioblastoma cells. Neither cell line expressed the 67 kDa form ofc-myc, as has been reported for other tumor cells and cell lines.

To assess the ability of endogenous c-myc to activate thepromoter/enhancer constructs shown in FIG. 11, each construct wasligated into the pCAT3Basic vector and aliquots of SJ-G2 cells weretransiently transfected with each of the plasmids. Results were reportedas CAT activity normalized to β-galactosidase activity, with activityproduced by the unmodified ODC promoter sequence arbitrarily set equalto 1.0.

The endogenous ODC promoter increased CAT activity ˜3-fold relative tocontrols with no ODC promoter. Four additional CACGTG E box sequences 5′to the endogenous ODC promoter (R4ODC) increased promoter activity7.2-fold relative to the unmodified promoter. Four additional CACGTG Ebox sequences 3′ to the ODC promoter or both 5′ and 3′ to the endogenouspromoter produced CAT activity similar to the unmodified promoter. Thehighest level of CAT activity, 14-fold greater than the ODC promoter,˜50-fold compared to promoterless controls, was produced by constructscontaining six additional CACGTG E box sequences 5′ to the ODC promoter(R6ODC). The negative control comprising the ΔR6ΔODC sequence gaveresults equivalent to controls lacking a promoter. SJ-G3 cells, which donot have immunodetectable c-myc, expressed only background levels of CATactivity when transfected with plasmids that contained either the mostefficient R6ODC sequence or the ΔR6ΔODC negative control sequence. Thesedata demonstrate that the R6ODC enhancer/promoter is the most efficientof the constructs tested in regulating expression of a reporter enzymein the SJ-G2 glioblastoma cell line that overexpresses c-myc.Accordingly, this promoter is particularly useful in the presentinvention in expression vectors for carboxylesterases.

The cDNA of rabbit liver CE was isolated by synthesizing degenerateoligonucleotides from amino acid residues 1-5 (SEQ ID NO:6) and 518-524(SEQ ID NO:10) of a published rabbit CE protein sequence (Korza, G. andJ. Ozols. 1988. J. Biol. Chem. 263:3486-3495). The oligonucleotidesconstructed are shown in FIG. 2. To amplify the rabbit cDNA by PCR, cDNAwas prepared from rabbit liver poly A+ mRNA and multiple samples wereprepared that contained the combination of oligonucleotide primers.Using PCR techniques, a single product was obtained from one set ofreactions that upon DNA sequencing was shown to encode the rabbit CE.

Since this represented a partial cDNA, both 5′ and 3′ RACE were used toamplify the entire coding sequence. Unique primers were designed fromthe partial DNA sequence. These oligonucleotides were used incombination with the AP1 primer to amplify sequences prepared fromMarathon adapted rabbit liver CDNA. Touchdown PCR (Don, R. H. et al.1991. Nucleic Acids Res. 19:4008) was performed in accordance with theMarathon cDNA amplification protocol.

The complete sequence of the CDNA (SEQ ID NO:20) and the derived aminoacid sequence (SEQ ID NO:21) of a rabbit liver CE are shown in FIG. 4.Northern analysis of the poly A+ mRNA from the rabbit liver with a[³²P]-labeled cDNA confirmed the presence of a single transcript ofapproximately 1.84 knt. No cross reaction was observed with any otherMRNA, consistent with this cDNA representing a unique RNA species.

Further, comparison of the amino acid sequence of the polypeptideencoded by the cDNA of the present invention with the published aminoacid sequence for rabbit CE (Swissprot Accession Number P12337; Korza,G. and J. Ozols. 1988. J. Biol. Chem. 263:3486-3495) showed threemismatches. In addition, the polypeptide encoded by the cDNA of thepresent invention contains an 8 amino acid insert and an 18 amino acidleader sequence at the N-terminus which the published sequence does notcontain. Accordingly, another aspect of the present invention relates tonovel polypeptides encoded by polynucleotides of the present invention.By “polypeptide” it is meant to include the amino acid sequence of SEQID NO: 21 depicted in FIG. 4 and fragments, derivatives and analogswhich retain essentially the same biological activity and/or function asthis rabbit liver CE.

The rabbit cDNA was expressed in bacteria. The 1.7 kb cDNA was ligatedinto pET32b and transformed into E. coli L21(DE3). Two clones wereisolated containing the rabbit cDNA either in the correct (pETRABFL) orincorrect (pETLFBAR) orientation with respect to the T7 promoter.Following induction of expression in liquid culture with IPTG, cellextracts were analyzed by SDS-PAGE and Western blotting. A 75 kDaprotein resulted from the fusion of the rabbit CE with the thioredoxinprotein in pETRABFL. Western analysis with the rat liver microsomal CEantibody and horseradish peroxidase (HRP)-conjugated protein S confirmedthat the 75 kDa protein encoded by PETRABFL contained the rabbit CE.Since other CEs are located in the ER and the primary sequence of therabbit enzyme contains similar characteristic leader and anchorsequences (Satoh, T. and M. Hosokawa. 1995. Toxicol. Lett.82/83:439-445), it is likely that the compartmentalization of the CE tothe ER is required for enzymatic activity. Indeed, overexpression of thehuman alveolar macrophage CE in E. coli failed to generate CE activity,however transfection of mammalian cells with the same cDNA yieldedsignificant conversion of o-NPA by whole cell extracts. In addition, therabbit CE demonstrated greater than 85% homology with human alveolarmacrophage CE yet the latter enzyme failed to convert CPT-11 to SN-38 inmammalian cells. This indicates that while CEs may have a broad range ofsubstrate specificities, the efficiency with which similar enzymeswithin different species can utilize a particular substrate variesdramatically.

To confirm that the cDNA encoded CE, the 1.7 kb EcoRI fragment wasligated into pCIneo to generate pCIRABFL and the plasmid transientlytransfected into Cos7 cells. pCIneo contains the SV40 origin ofreplication allowing plasmid amplification in cells expressing the largeT antigen, such as Cos7. The IC₅₀ value for CPT-11 for cells expressingthe CE was approximately 8-80 fold, and most typically about 56 fold,less than that of the parent cell line thus indicating that the enzymehas sensitized mammalian cells to CPT-11 (see FIG. 5).

Rabbit CE has also been expressed in Spodoptera frugiperda S21 cells viaa baculovirus vector. CE secreted in these cells was concentrated byultrafiltration to approximately 1 ml containing approximately 30,000micromoles/millimeter of enzyme activity.

Experiments were also performed to determine whether human CE fromsources other than liver were capable of converting CPT-11 to its activemetabolite. Mouse small intestine is known to express high levels of CEthat can convert CPT-11 to SN-38. Accordingly, the ability of humanintestinal CE (hiCE) as an activator of CPT-11 was examined. Using humanintestinal mucosal biopsy tissue, the conversion of o-NPA to nitrophenolby whole tissue sonicates was monitored. CE activity was identified inboth small intestine and colon samples, with activity levels in smallintestine being much less than the levels seen with human liver.However, the amount of CPT-11 conversion was essentially the same on amg protein basis (Table 1). Thus, these data indicate that for the totallevels of esterases present in tissue, the percentage of CPT-11converting enzymes in the small intestine is greater than the percentagein the liver.

TABLE 1 Metabolism of o-NPA and CPT-11 by Human Biopsy Extracts o-NPAConversion CPT-11 Conversion Sample (μmoles/min/mg) (pmoles/hr/mg) Smallintestine 113.0 ± 9.2  7.57 Small intestine 67.6 ± 4.1  3.13 Smallintestine 61.0 ± 2.0  3.83 Colon 75.9 ± 2.6  1.34 Colon 46.5 ± 1.2  0.65Colon 86.3 ± 5.2  2.06 Liver 1928.9 ± 251.0  7.15 Liver 802.7 ± 68.2 2.72

A cDNA encoding a human intestinal CE has been isolated (Schwer et al.1997 Biochem. Biophys. Res. Commun. 233(1):117-120) and shown to bepredominately expressed in the small intestine. To determine whether theisolated enzyme was capable of activation of CPT-11, the full lengthcoding sequence of the human intestinal CE (GenBank Accession No.Y09616) was obtained by PCR using oligonucleotide primers that createdXbaI restriction sites adjacent to the ATG initiation and TAGtermination codons. The cDNA (SEQ ID NO:27) was amplified from humanliver cDNA (Clontech, Palo Alto, Calif.) using Taq polymerase. Productswere then ligated into pCR-II TOPO and sequenced to verify theiridentity. One clone containing the bona fide sequence was ligated intopCEneo (pClhiCE) for expression in mammalian cells. Sequence analysisindicated that the rabbit CE demonstrates 81% identity with human liverCE but only 47% identity with hiCE. In addition, human liver CEdemonstrated 49% identity with hiCE.

Accordingly, sequence similarity does not predict the ability of a CEenzyme to metabolize CPT-11. Instead, computer modeling studies indicatethe ability of a CE to activate CPT-11 is dependent on the residues thatform the entrance to the active site gorge of these proteins. Thus, itis expected that other CEs with residues similar to those forming theentrance to the active site gorge in rabbit liver CE and humanintestinal CE will also be useful in metabolizing chemotherapeuticprodrugs and inactive metabolites thereof, such as CPT-11 and APC,respectively, to active drug.

Using sonicates of cells expressing hiCE, experiments showed that therewas efficient conversion of both o-NPA and CPT-11. No CE activity orCPT-11 conversion was detected in media of cells transfected with hiCEindicating that the protein was not secreted from cells (Table 2).

TABLE 2 Conversion of o-NPA and CPT-11 by COS-7 Cells o-NPA ConversionCPT-11 Conversion Plasmid Enzyme (μmoles/min/mg) (pmoles/hr/mg) pCIneonone  6.7 ± 0.15 3.4 pCIneo¹ media 13.3 ± 2.7  0.9 pCIhiCE hiCE 1735.6 ±163.1  654.3 pCIhiCE¹ media 30.9 ± 2.8  2.1 pCIHUMCAR hCE1 4780.3 ±279.8  9.6 pCIRAB rabbit 2755.5 ± 271.2  2323.0

Another aspect of the present invention relates to the ability ofcompositions comprising a polynucleotide encoding a carboxylesterase anda disease-specific responsive promoter of selected tumor cells tosensitize the tumor cells to a chemotherapeutic prodrug. The ability ofa rabbit CE or a human intestinal CE of the present invention tosensitize human tumor cells to CPT-11 was examined. Experiments werefirst performed to confirm that the metabolite produced by the activityof a CE of the present invention is biologically active in vitro. Rh30cells were exposed to the products of each reaction for one hour and thepercentage of growth inhibition was determined. As expected, Rh31 cellsexposed to 1 to 5 units of CE that had been inactivated by heatingproduced no inhibition of cell growth. In contrast, reaction products ofCPT-11 incubated with 1 to 5 units of active CE produced a 30-60%inhibition of cell growth. These data are consistent with the conversionof CPT-11 to SN-38 by CE in these cells. Similar confirmatoryexperiments were performed with COS-7 cells.

The CE activity of extracts of the transfected cells was thendetermined. First, the IC₅₀ values for CPT-11 in Rh30 rhabdomyosarcomacells that had been stably transfected with a rabbit liver CE cDNA ofthe present invention or the pIRES vector alone were also determined.Cells transfected with the CE CDNA contained approximately 60-fold moreCE activity than control cells. The IC₅₀ of CPT-11 for Rh30pIRES cells(no CE cDNA) was 4.33×10⁻⁶ M while the IC₅₀ for the Rh30pIRES_(rabbit)cells was 5.76×10⁻⁷ M. Therefore, the transfected cells were more than8-fold more sensitive to CPT-11. These data are consistent with anincreased conversion of CPT-11 to SN-38 in the cells transfected with aCE of the present invention.

To determine whether the human intestinal CE could confer similarsensitivity to CPT-11, the effect of the drug on growth of COS-7 cellsexpressing hiCE was examined. The IC₅₀ of cells expressing hiCE was 0.5μM, approximately 11-fold less than that of cells transfected with theparent plasmid (pCEneo IC₅₀=5.4 μM). These data indicate that efficientin vivo activation of CPT-11 by hiCE also occurred leading to asensitization of cells to the drug.

Experiments have also been conducted which demonstrate that a CE of thepresent invention is capable of converting the inactive metabolite APCto SN-38. Structures of these compounds are shown in FIG. 8. FIG. 6shows the results of experiments in vitro where APC is converted toSN-38 in a concentration-dependent manner by a rabbit CE of the presentinvention. These data confirm the unique ability of a CE of the presentinvention to activate the prodrug CPT-11, as well as to activate one ofits metabolites. Further, experiments in U-373 cells that express a CEof the present invention showed that these cells were sensitized to thegrowth inhibitory effects of APC (see FIG. 7).

In vivo efficacy of the CE of the present invention to sensitize tumorcells to CPT-11 has also been demonstrated in two different types oftumor cells. Experiments conducted in a mouse model demonstrate that aCE of the present invention is capable of sensitizing cells to thegrowth inhibitory effects of CPT-11.

In a first set of experiments, the ability of rabbit CE to sensitizeRh30 rhabdomyosarcoma human tumor cells grown as xenografts inimmune-deprived mice was demonstrated. In this preclinical model,expression of the transfected cDNA for rabbit CE was maintained for atleast 12 weeks. Importantly, tumors were advanced (greater than 1 cm³ involume) before treatment with CPT-11 began. As depicted in FIG. 9B,tumors in mice expressing CE and treated with 2.5 mg CPT-11/kg/day forfive days each week for two weeks (one cycle of therapy), repeated every21 days for a total of three cycles (over 8 weeks), regressed completelyand did not regrow during the 12 weeks of the study. In contrast, tumorsthat did not express the CE regressed only transiently with CPT-11treatment, with regrowth occurring within one week after CPT-11treatment stopped (see FIG. 9C).

In a second set of experiments, human U373 glioblastoma xenografts thatexpress rabbit liver CE were shown to be more sensitive to CPT-11 thanxenografts transfected with a control plasmid (no rabbit CE). Xenograftsestablished from cells transfected with the plasmid encoding rabbit CEregressed completely while xenografts from cells transfected with thecontrol plasmid showed stable disease but no significant regression (seeFIG. 10).

Thus, these data support the use of the combination of polynucleotideencoding a CE of the present invention and CPT-11 to reduce the amountof CPT-11 needed to produce inhibition of tumor cell growth, or tosensitize the tumor cells to CPT-11. These data also support the use ofthe present invention to allow for decreased dosage with CPT-11 incancer patients, thus reducing the likelihood of dose-limiting toxicity.Further, as shown by these experiments, APC, which is relativelynontoxic, can also be used as a chemotherapeutic prodrug in combinationwith a CE of the present invention to produce tumor-specific cell deathwhile minimizing toxic side effects.

The present invention thus also relates to a method for treating cancerwith reduced side effects. In one embodiment, a polynucleotide of thepresent invention is inserted into a viral vector using a gene transferprocedure. Preferred viral vectors include, but are not limited to,retroviral, adenoviral, herpesvirus, vaccinia viral and adeno-associatedviral vectors. In this embodiment, it is preferred that the vectorfurther comprise a disease-specific responsive promoter. The vectors canthen be injected into the site of tumor removal along with systemicadministration of a prodrug such as CPT-11 to inhibit the recurrence oftumors due to residual tumor cells present after surgical resection of atumor.

Alternatively, the viral vector can be used to purge bone marrow ofcontaminating tumor cells during autologous transplant. Bone marrowpurging via a viral vector such as adenovirus which expresses a CE ofthe present invention is performed ex vivo. Efficiency of removal ofcontaminating tumor cells is determined by PCR assays of purged samples.Data indicate that the method of the present invention is applicable toan animal model for purging bone marrow of neuroblastoma cells such asthat described in Example 6. Methods for preparation of the vectors,modes of administration, and appropriate doses of prodrug are well knownto those of skill in the art. Other methods of gene delivery such aschemical and liposome-mediated gene transfer, receptor-mediated DNAuptake, and physical transfer by gene guns or electroporation may alsobe employed.

Another method for delivering CEs to selected tumor cells involvesantibody direct enzyme prodrug therapy (ADEPT). In this method, humantumors are targeted by conjugation of tumor-specific marker antibodywith a molecule such as rabbit liver CE. Cellular internalization of thecomplex and release of active CE would be achieved, leading to CPT-11activation that is specific for cells expressing the marker antigen.Since the array of marker molecules expressed upon the cell surface isdifferent for each tumor type, markers specific for each targeted tumortype can be selected as appropriate. Similarly, the use of avidin-biotinconjugated molecules to target tumor cells (Moro, M. et al. 1997. CancerRes. 57:1922-1928) is also applicable for localization of CEs to thecell surface followed by drug activation at the targeted cell.

The rabbit liver CE is localized in the endoplasmic reticulum. Removalof the six terminal amino acids results in secretion of active proteininto the extracellular milieu. Both the secreted and the endoplasmicreticulum-localized protein can convert CPT-11 to SN-38; therefore, thepotential exists for a bystander effect from cells expressing thesecreted enzyme. A similar bystander effect has been demonstrated forother enzyme/prodrug combinations, such as HSVtk and ganciclovir(Dilber, M. S. et al. 1997. Cancer Res. 57:1523-1528), and results inincreased cytotoxicity. Extracellular activation of CPT-11 may result inmore efficient eradication of MRD in that uninfected neighboring tumorcells would be killed by exogenously produced SN-38. Gene therapyprotocols with a secreted CE in combination with CPT-11 may therefore bemore appropriate for the elimination of residual tumor tissue.Accordingly, in this embodiment, it may be preferred to use a fragmentof a polynucleotide encoding a polypeptide which is secreted. Forexample, for rabbit liver, a cDNA encoding a protein which does notcontain the six terminal amino acids depicted in FIG. 4, or a cDNAencoding a rabbit liver CE enzyme consisting of amino acids 1-543 (SEQID NO:26) of FIG. 4, may be preferred. Additionally, recent reportsindicate that the tethering of drug activating enzymes to theextracellular cell surface can result in anti-tumor activity in humantumor xenografts when combined with appropriate prodrug (Marais, R. etal. 1997. Nature Biotech. 15:1373-1377). A tethered enzyme generates alocal bystander effect since the protein is not free to circulate in theplasma. Attachment of a CE of the present invention to the cell surfaceshould result in local extracellular activation of CPT-11 to SN-38 andenhance local cell kill. Purging bone marrow of contaminating tumorcells will be accomplished by an intracellular enzyme, whereaseradication of MRD is better achieved by an enzyme that activates CPT-11at an extracellular location.

CEs of the present invention cleave the COOC bond present as an esterlinkage in CPT-11 to generate SN-38 (see FIG. 8). Since these enzymesmay also catalyze the activation of other compounds that contain such alinkage, the present invention also provides assays for screening forcompounds that contain this and related moieties. In one embodiment, theassay of the present invention is conducted in a cell system using, forexample, yeast, baculovirus, or human tumor cell lines. In thisembodiment, compounds activated by CE will be identified and assessedfor anticancer activity by growth inhibition or clonogenic cell survivalassays using cells expressing or lacking a CE of the present invention.Alternatively, compounds can be screened in cell-free assays using a CEof the present invention isolated from host cells expressing thisenzyme. In this embodiment, the ability of the enzyme to cleave a COOCester linkage of a candidate compound is measured directly in a standardenzyme assay buffer system containing a CE of the present invention.Known concentrations of candidate compounds can be added to assay tubescontaining a biological buffer such as HEPES at pH 7.4 and the enzyme,and incubated at 37° C. for a selected amount of time. The reaction isthen terminated by addition of methanol. Following termination of thereaction, the assay tubes are centrifuged and the supernatant analyzedfor the presence of cleaved compound fragment. Analysis of thesupernatant can be performed by any number of well known techniquesincluding, but not limited to, spectrofluorometric analysis, highpressure liquid chromatography or mass spectrometry. Compoundsidentified in these screening assays as potential anticancer prodrugsmay require chemical modification for optimize their anti-tumoractivity.

The following non-limiting examples are provided to further illustratethe claimed invention.

EXAMPLES Example 1 Identification of CEs

A CE enzyme suitable for converting CPT-11 to the active form, SN-38 wasidentified by testing a variety of samples. This screening includedenzymes from a series of sera, cell extracts and commercially availableCEs using a rapid fluorometric assay. Certain of these enzymes showactivity in metabolism of CPT-11.

Since partially purified CEs were commercially available, several ofthese were also tested for their ability to metabolize CPT-11. Bothrabbit and pig liver CEs metabolized CPT-11 efficiently. Thecommercially available pig CE contained several proteins. However, themajor bands were very similar in molecular weight and did not separateusing SDS-PAGE. In contrast, the rabbit preparation consisted of onlyone major and one minor protein. Therefore, the rabbit proteins werechosen for further study.

The rabbit proteins were subjected to automated N-terminal amino acidsequencing. Both bands yielded protein sequences indicating that thepeptides were not N-terminally blocked. The derived amino acid sequenceswere analyzed by computer searches using the Fasta and BLAST comparisonprograms. Band 1 (approximately 60 kDa) demonstrated significanthomology with several CE sequences, including a rabbit CE, present inthe GenBank and Swissprot databases (FIG. 1). However, the nucleic acidsequence encoding rabbit CE protein has not been disclosed. In addition,comparison of the amino acid sequence of the polypeptide encoded by thecDNA of the present invention with the published amino acid sequence forrabbit CE showed three mismatches. Further, the polypeptide encoded bythe CDNA of the present invention contains an 8 amino acid insert and an18 amino acid leader sequence at the N-terminus which the publishedsequence does not contain. Thus, the published amino acid sequence of arabbit liver carboxylesterase protein (Swissprot Accession NumberP12337; Korza, G. and J. Ozols. 1988. J. Biol. Chem. 263:3486-3495) isdifferent from the polypeptide encoded by the cDNA of the presentinvention.

In addition to the rabbit CE, studies were performed to isolate human CEfrom sources other than liver, since the human liver CE has been shownto be an inefficient enzyme for metabolism of CPT-11. Biopsies of humanintestine were obtained from the Cooperative Human Tissue Network(Birmingham, Ala.). The samples were ground under liquid nitrogen andthe resulting powder sonicated in 50 mM Hepes, pH 7.4, on ice. CEactivity and CPT-11 conversion were monitored by these extracts.

Example 2 Cloning of Rabbit Carboxylesterase

The cDNA encoding the rabbit CE protein of the present invention wasisolated by synthesizing degenerate oligonucleotides from amino acidresidues 1-5 (SEQ ID NO:6) and 518-524 (SEQ ID NO:10) of the publishedprotein sequence of a rabbit liver CE (Korza, G. and J. Ozols. 1988. J.Biol. Chem. 263:3486-3495). The oligonucleotides constructed are shownin FIG. 2. To amplify the rabbit cDNA by PCR, cDNA was prepared fromrabbit liver poly A+ MRNA and multiple samples were prepared thatcontained the combination of oligonucleotide primers. Following heatingat 95° C. for five minutes, the polymerase was added at the annealingtemperature and reactions cycled as follows: 94° C. 45 seconds,annealing temperature (46-58° C.) 1 minute, 72° C. 90 seconds.Typically, 25 cycles of amplification were performed. A single productwas obtained from one set of reactions that upon DNA sequencing wasshown to encode a novel rabbit CE.

Since this represented a partial CDNA, both 5′ and 3′ RACE were used toamplify the entire coding sequence. Unique primers of 27 and 28nucleotides, corresponding to the 5′ and 3′ ends respectively, weredesigned from the partial DNA sequence. These oligonucleotides were usedin combination with the AP1 primer to amplify sequences prepared fromMarathon adapted rabbit liver cDNA. Touchdown PCR (Don, R. H. et al.1991. Nucleic Acids Res. 19:4008) was performed as according to theMarathon cDNA amplification protocol. A single product of approximately420 bp was generated by the 3′ primer, however no product was observedwith the 5′ oligonucleotide. Standard PCR amplification protocols (94°C. 45 seconds, 60° C. 1 minute, 72° C. 1 minute, 30 cycles) resulted ina smear of DNA products with a minor band at approximately 280 bp.Attempts to increase the specificity of the reaction were unsuccessful.Therefore, DNA was isolated from the agarose gels and then ligated intopCRII-TOPO. DNA sequencing indicated the presence of the oligonucleotideRACE primers in both samples. The 3′ RACE product extended 407 bp fromthe specific primer and encoded the terminal amino acids consistent withthe published data (Korza, G. and J. Ozols. 1988. J. Biol. Chem.263:3486-3495). In addition, a poly A tail was present and the originalMarathon cDNA synthesis primer sequences could be identified. The 5′RACE product extended 247 bp from the CE specific primer and encoded thepublished amino acid sequence. An additional 18 residue hydrophobicleader sequence beginning with a methionine initiation codon wasidentified, consistent with the amino acids present at the N-termini ofCEs derived from other species (FIG. 3). The entire transcript includingboth untranslated 5′ and 3′ sequences, as determined by the RACEexperiments, was 1886 nt long, very similar to that indicated by theNorthern analysis. This confirmed that the cDNA described in theseexperiments was full length.

To amplify a full length rabbit CE CDNA, oligonucleotide primersRabNTERM (GGCAGGAATTCTGCCATGTGGCTCTG; SEQ ID NO:23) and RabCTERM(CGGGAATTCACATTCACAGCTCAATGT; SEQ ID NO:24) were designed to createEcoRI sites 9 bp upstream of the ATG initiation codon and 8 bpdownstream of the TGA termination codon. These were used to amplifyrabbit liver cDNA using Pfu polymerase. The initial 5 cycles ofamplification were performed as follows: 94° C., 45 seconds; 50° C., 1minute; 72° C., 90 seconds with the annealing temperature raised to 56°C. for the subsequent 25 cycles. This allowed the formation of the EcoRIrestriction sites at the termini of the cDNA. A product of approximately1700 bp was obtained, ligated into pUC9 restricted with EcoRI and theentire DNA was sequenced.

Example 3 Expression of Rabbit CE in Spodoptera frugiperda Sf21

Cells (4×10⁷) were plated in the lower chamber of an Integra CL1000flask (Integra Biosciences, Ijamsville, Md.) in 45 mls of Insect Xpressmedia (Biowhittaker, Walkersville, Md.). To ensure adequate growth ofthe cells, 500 mls of complete Grace's media was added to the upperchamber of the flask. After incubation at 27° C. for 2 days, baculoviruswere added to the cells in the lower chamber at a multiplicity ofinfection of 20. Media in the lower chamber was assayed every 24 hoursfor carboxylesterase (CE) activity and usually harvested after 120hours. The secreted CE was concentrated by ultrafiltration to yieldapproximately 1 ml of sample containing approximately 30,000micromoles/ml of enzyme activity.

Example 4 Amplification of Human Intestinal CB cDNA

The full length coding sequence of the human intestinal CE (GenBankAccession No. Y09616) was obtained by PCR using oligonucleotide primersHumICE3′ (CGGTCTAGAGAGCTACAGCTCTGTGTGTCTG; SEQ ID NO:29) and HumICE5′(CGAGTCTAGAGAGCCGACCATGCGGCTGCAC; SEQ ID NO:30) that created XbaIrestriction sites adjacent to the ATG initiation and TAG terminationcodons. The cDNA was amplified from human liver cDNA (Clontech, PaloAlto, Calif.) using Taq polymerase under the following conditions;denaturation at 94° C., 45 seconds, annealing at 50° C., 1 minute, andextension at 72° C., 2 minutes. Following 30 cycles of amplification,products were ligated into pCR-II TOPO and sequenced to verify theiridentity. One clone containing the bona fide sequence was ligated intopCIneo (pCIhiCE) for expression in mammalian cells. Plasmids containingthe human liver CE (hCE1; PCIHUMCAR) and the rabbit liver CE (pCIRAB)have been previously described (Potter et al. 1998. Cancer Res.52:2646-2651; Potter et al. 1998. Cancer Res. 58:3627-3632).

Example 5 Transfection of COS-7 Cells with Human Intestinal CE

COS-7 cells were transfected by electroporation as previously described(Potter et al. 1998 Cancer Res 52:2646-2651). Extracts were prepared bysonication of cell pellets in minimal volumes of 50 mM HEPES (pH 7.4) onice 48 hours following transfection.

Esterase Assays

Esterase activity was determined in whole tissue sonicates using aspectrophotometric assay with o-nitrophenol acetate as a substrate(Potter et al. 1998 Cancer Res. 52:2646-2651; Beaufay et al. 1974 J.Cell Biol. 61:188-200). Protein concentrations were calculated usingBioRad protein assay reagent (Hercules, Calif.) with bovine serumalbumin as a standard. Enzyme activities were calculated as μmoles ofo-nitrophenol produced per minute per mg of total protein.

Transfection of Mammalian Cells

COS-7 cells (10⁷) were electroporated with 20 μg of plasmid DNA in avolume of 200 μl of phosphate buffered saline using a Bioradelectroporator and a capacitance extender (Biorad, Hercules, Calif.).Optimized conditions for electroporation were achieved using 260 V and960 μF. Following transfection, cells were plated into 75 cm² flasks infresh media and harvested by trypsinization after 48 hours.

CPT-11 Conversion Assays

Appropriate amounts of extracts were incubated with 5 μM of CPT-11 in afinal volume of 200 μl of 50 mM HEPES pH 7.4 at 37° C. for 24 hours.Reactions were terminated by addition of 200 μl cold acid-methanol andcentrifuged for 15 minutes at 16000 g. The conversion of CPT-11 to SN-38was monitored by high performance liquid chromatography (HPLC) in 20 μlsample volumes.

Example 6 In vitro Biological Activity of Rabbit CE

The in vitro activity of rabbit liver CE was examined in tumor celllines. The growth inhibition of CPT-11 was compared in cells with andwithout active rabbit CE. The cells used were Rh30 cells (10⁷) that hadbeen electroporated with 20 μg of IRES plasmid DNA or plasmid containingCE cDNA in a volume of 200 μl of phosphate buffered saline. Optimizedconditions for electroporation were achieved using 180 V and 960 μF. Thecells were plated into 75 cm² flasks in fresh media and 500 μg G418/mladded 48 hours following transfection to select for cells expressing theneo gene and the CE. Cells were grown for a minimum of 10 days beforeuse in growth inhibition experiments.

In the first assay, CPT-11 was pre-incubated with rabbit liver CE toproduce SN-38 prior to exposure of the cells to drug. Specifically, 0.5to 5 units of CE were incubated with 1 μM CPT-11 at 37° C. in DMEMmedium for 2 hours. Each reaction mixture was then filter-sterilized andRh30 cells were exposed to drug for one hour, at which time the mediumwas replaced with drug-free medium containing serum. Enzyme that hadbeen inactivated by boiling for five minutes prior to incubation withdrug or CPT-11 to which no enzyme had been added were used as negativecontrols. Cells were allowed to grow for 3 cell doubling times and cellnumbers were determined.

In the second type of growth inhibition assay, Rh30 cells that had beentransfected with either pIRES parent plasmid DNA or the plasmidcontaining the rabbit CE cDNA were exposed to different concentrationsof CPT-11. Drug was added to tissue culture medium of each of the stablytransfected cell lines for two hours, after which time the medium wasreplaced with drug-free medium. Cells were then allowed to grow for 3cell doublings as before. Results were expressed as the concentration ofdrug required to reduce cell growth to 50% of control cells, or IC₅₀.

Results showed that extracts of the transfected cells contained greaterthan 60-fold more CE activity than controls as determined by theconversion of o-nitrophenyl acetate to o-nitrophenol. Further, theRh30pIRES cells transfected with rabbit CE were greater than 8-fold moresensitive to CPT-11 than controls, as shown by a decrease in the IC₅₀values. Therefore, Rh30 cells stably transfected with rabbit CE weremore sensitive to growth inhibition by CPT-11 than cells that did notcontain the cDNA for rabbit CE.

Example 7 In vitro Biological Activity of Human Intestinal CE

CE activity was determined by the spectrophotometric method describedabove for rabbit CE samples using o-NPA as a substrate. In anotherassay, activation of CPT-11 was determined by incubating samples of hiCEwith either 5 μM or 25 μM CPT-11 in a total volume of 200 μl of 50 mMHepes pH 7.4 at 37° C. for up to 20 hours. Reactions were terminated bythe addition of an equal volume of cold acidified methanol, followed bycentrifugation at 100,000×g for 30 minutes. The levels of SN-38 producedin the reaction were quantitated by HPLC.

Growth inhibition assays were performed with COS-7 cells as previouslydescribed (Potter et al. 1998 Cancer Res. 52:2646-2651; Danks et al.1998 Cancer Res. 52:2646-2651, 1998). Forty-eight hours aftertransfection, 5×10⁴ cells were plated into 1.5 cm diameter dishes andallowed to attach overnight. CPT-11 diluted in fresh medium was appliedfor two hours and the cells allowed to grow for three days, equivalentto three cell doublings. Cell number was determined by counting using aCoulter Multisyser II (Coulter Electronics, Luton, England) and growthinhibition curves were plotted using Prism software (GraphPad SoftwareInc., San Diego, Calif.). IC₅₀ values (the concentration of drugrequired to reduce cell growth by 50%) were calculated from these curvefits.

Example 8 Rabbit CE Activates APC, a Novel Prodrug

In addition to efficiently converting CPT-11 to the active compoundSN-38, experiments were also performed demonstrating the ability ofrabbit liver CE to convert the inactive metabolic end product APC toSN-38. No known human enzyme activates APC. FIG. 6 shows the kinetics ofconversion of APC to SN-38 by 50 units of rabbit liver CE in an in vitroreaction. FIG. 7 shows that U-373 glioma cells that express the rabbitliver CE, but not human alveolar macrophage carboxylesterase which is85% homologous to the rabbit enzyme, are sensitized to the growthinhibitory effects of APC. Thus, the combination of APC andsensitization of selected tumor cells with rabbit liver CE as describedabove can be used to produce a tumor-specific cell death while greatlyminimizing the toxic side effects associated with administration ofchemotherapy.

Example 9 Use of Rabbit CE in an In vivo Model for MRD

A xenograft model for MRD has been developed to demonstrate theeffectiveness of the combination of rabbit CE and prodrug in theprevention of MRD. In this model, treatment of immune-deprived mice,i.e., SCID mice, bearing human NB-1691 xenografts with 10 mg/kg CPT-11daily for 5 days on two consecutive weeks results in complete regressionof the tumor. However, within 4-6 weeks, tumors are palpable in theexact position where the original xenograft was implanted. Since thesetumors arise from cells that survived the initial cycle of chemotherapy,this model therefore mimics results seen in patients following surgicalresection of the primary tumor and subsequent regrowth at the same site.

Experiments were performed in this model to compare the responses ofmice bearing human Rh30 and Rh30pIRES_(rabbit) xenografts. Rh30rhabdosarcoma xenografts were transfected with pIRESneo plasmidcontaining the cDNA for rabbit liver CE and selected with G418.Expression of CE was confirmed by biochemical assay using the CEsubstrate o-NPA and maintained for at least 12 weeks. Two groups of SCIDmice were then injected with the transfected Rh30pIRES_(rabbit) cellssubcutaneously into the flanks. A third group of control mice wasinjected in identical fashion with Rh30 cells not transfected with theplasmid. When the tumors reached a size of approximately 1 cm³, 2.5 mgCPT-11/kg/day was administered five days each week for two weeks (onecycle of therapy), repeated every 21 days for a total of three cycles(over 8 weeks) to one group of mice injected with the transfectedRh30pIRES_(rabbit) cells and the third group of control mice.

The tumors expressing rabbit CE regressed completely and did not regrowduring the 12 weeks of the study (FIG. 9B). In contrast, tumors notexpressing the CE regressed only transiently, regrowing within one weekafter CPT-11 treatment had stopped (FIG. 9C).

Similar studies were performed employing U373 glioblastoma cellstransfected with the pIRESneo plasmid or with pIRESneo containing thecDNA for rabbit liver CE and selected with G418. Expression of CE in thetumor cells was confirmed by biochemical assay using the substrateo-NPA. Cells were injected subcutaneously into the flanks of the SCIDmice. When tumors reached approximately 1 cm³ in size, CPT-11 wasadministered daily for five days each week as described above, for threecycles, at a dose of 7.5 mg/kg/day.

The U373 cells that expressed rabbit CE were also more sensitive toCPT-11. Xenografts established from cells transfected with the plasmidencoding rabbit CE regressed completely while xenografts from cellstransfected with the control plasmid showed stable disease with nosignificant regression. These data in two different human tumorxenografts demonstrate the in vivo efficacy of rabbit CE to sensitizetumor cells to CPT-11.

Similar experiments can be performed to assess the in vivo efficacy ofhiCE in preventing MRD. In these experiments, adenovirus expressing hiCEunder control of a tumor-specific promoter is administeredsubcutaneously at the site of xenograft implantation in this modelduring the 4 to 6 week period when tumors are not present, followed bytreatment with low doses of CPT-11. Typically, since tumor regression iscomplete 3 weeks after commencing treatment with CPT-11, adenovirus/drugadministration begins at week 4. In initial experiments, adenovirus isadministered on Monday, Wednesday, Friday and CPT-11 is given daily onTuesday through Saturday for two cycles. This permits determination ofthe most tolerated, effective schedule and dosage of adenovirus andCPT-11 administration to produce the longest delay of recurrent disease.These results are used to determine correct dosage for treatment ofhuman MRD. The starting point for the animal experiments is injection of10⁵ to ₁₀ ⁸ pfu of adenovirus containing the hiCE of the presentinvention.

Example 10 Use of a CE/Prodrug Combination to Purge Bone Marrow of TumorCells

Intravenous injection of human neuroblastoma NB-1691 tumor cells intoimmune-deprived mice results in the development of widespread metastaticdisease with death occurring on days 36-38. Since both synaptophysin andtyrosine hydroxylase expression are specific for neuroblastoma cells,RT/PCR analysis of these mRNAs can detect tumor cells present in mixedpopulations of cells. Circulating neuroblastoma cells can be detected inthe peripheral blood of these animals 36 days after injection withNB-1691. Studies will then determine whether the bone marrow of thesesame animals contains neuroblastoma cells. The success of ex vivopurging of bone marrow with the rabbit liver CE/CPT-11 combination orthe human intestinal CE/CPT-11 combination is demonstrated bytransplanting purged bone marrow into lethally irradiated mice. If miceremain disease free for extended periods of time, this indicates thatthe adenoviral CE/prodrug purging therapy kills neuroblastoma cells inthe donor marrow.

Example 11 Treatment of Minimal Residual Disease (MRD) in Humans

The rabbit CE or human intestinal CE in combination with CPT-11 or otherprodrugs activated by this enzyme is used to purge bone marrow ofresidual tumor cells prior to autologous bone marrow transplants toprevent recurrence of local MRD following removal of bulk tumor bysurgery or chemotherapy. Following debulking of the primary tumor,adenovirus containing the rabbit liver CE or human intestinal CE underthe control of a tumor-responsive promoter is applied to the tumormargins at either the time of surgery, by stereotaxic injection, or byimplantation of a time-release polymer or other material. Anti-tumoreffect of single application at time of surgery is compared with theeffect produced by repetitive or time-release use of adenoviralconstructs. Adenovirus dose ranges from 10⁶ to 10¹⁰ plaque-forming unitsas has been reported to be effective for intratumoral injection ofadenovirus (Heise, C. et al. 1977. Nature Med. 3:639-645). CPT-11 isadministered over the next one to six weeks to elicit tumor selectivecell kill. Doses and schedules of CPT-11 are determined in clinicaltrials of CPT-11 by itself and in human xenograft model systems toproduce maximal tumor effect.

Example 12 Purging Bone Marrow of Tumor Cells in Humans

Tumor cells that contaminate bone marrow used for autologous transplantcontribute to relapse of disease. Therefore, the rabbit liver CE or thehuman intestinal CE is used in combination with a suitable prodrug toeradicate tumor cells in marrow samples to be used for transplant. Thisapproach maintains the viability of hematopoietic cells required forreconstitution. Bone marrow samples are transduced ex vivo withadenovirus containing the rabbit liver CE cDNA or the human intestinalCE cDNA, using a multiplicity of infection (moi) that will infect 100%of the tumor cells. Typically, a moi of 0.5 to 10 is adequate for tumorcells, while a moi of 100 to 1,000 is required to transduce a majorityof hematopoietic progenitor cells. Two days following adenoviraltransduction, cells are exposed for two hours to a range of CPT-11concentrations, usually varying from 50 nM to 100 μM. Two days afterexposure to drug, the marrow sample is harvested and stored forreinfusion into the patient and reconstitution of a tumor-free marrow.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 30 <210> SEQ ID NO 1 <211>LENGTH: 34 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <221> NAME/KEY: UNSURE <222> LOCATION: (7) <221> NAME/KEY:UNSURE <222> LOCATION: (33) <221> NAME/KEY: UNSURE <222> LOCATION: (22)<223> OTHER INFORMATION: Synthetic <400> SEQUENCE: 1 His Pro Ser Ala ProVal Xaa Val Asp Thr Val His Gly Lys Val Leu 1 5 10 15 Gly Lys Phe ValSer Xaa Glu Gly Phe Ala Gln Pro Val Ala Lys Phe 20 25 30 Xaa Gly <210>SEQ ID NO 2 <211> LENGTH: 36 <212> TYPE: PRT <213> ORGANISM: Oryctolaguscuniculus <400> SEQUENCE: 2 His Pro Ser Ala Pro Pro Val Val Asp Thr ValLys Gly Lys Val Leu 1 5 10 15 Gly Lys Phe Val Ser Leu Glu Gly Phe AlaGln Pro Val Ala Val Phe 20 25 30 Leu Gly Val Pro 35 <210> SEQ ID NO 3<211> LENGTH: 54 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400>SEQUENCE: 3 Met Trp Leu Arg Ala Phe Ile Leu Ala Thr Leu Ser Ala Ser AlaAla 1 5 10 15 Trp Gly His Pro Ser Ser Pro Pro Val Val Asp Thr Val HisGly Lys 20 25 30 Val Leu 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7 cacccaagcg cacc 14 <210> SEQ IDNO 8 <211> LENGTH: 14 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Synthetic <221> NAME/KEY: unsure <222> LOCATION:(6) <221> NAME/KEY: unsure <222> LOCATION: (12) <400> SEQUENCE: 8cacccnagcg cncc 14 <210> SEQ ID NO 9 <211> LENGTH: 14 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic <221>NAME/KEY: unsure <222> LOCATION: (6) <221> NAME/KEY: unsure <222>LOCATION: (9) <221> NAME/KEY: unsure <222> LOCATION: (12) <400>SEQUENCE: 9 cacccntcng cncc 14 <210> SEQ ID NO 10 <211> LENGTH: 7 <212>TYPE: PRT <213> ORGANISM: Oryctolagus cuniculus <400> SEQUENCE: 10 AlaPhe Trp Thr Glu Leu Trp 1 5 <210> SEQ ID NO 11 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Oryctolagus cuniculus <400> SEQUENCE: 11gcattctgga cagaactatg g 21 <210> SEQ ID NO 12 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Oryctolagus 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gaattctgcc atgtggctct gtgcattggc cctggcctctctcgccgctt gcacggcttg 60 ggggcacccg tctgcaccac ctgtggtaga tactgtgcatggcaaagtcc tggggaagtt 120 cgtcagctta gaaggatttg cacagcccgt ggccgtcttcctgggagtcc ccttcgccaa 180 gccccctctt ggatccctga ggtttgcacc accacagcctgcagaatcat tgagccacgt 240 gaagaacacc acctcctacc ctcccatgtg ctcccaggacgcagtatcag ggcatatgct 300 ctcggagctc ttcaccaaca gaaaagagaa catccctcttaagttttctg aagactgcct 360 ttacctgaat atttacaccc ctgctgacct gacaaagagaggcaggctgc cggtgatggt 420 gtggatccat ggaggtggtc tgatggtggg tggagcatcaacctatgatg gcctggctct 480 ttctgcccat gagaacgtgg tggtggtgac cattcagtaccgcctgggca tctggggatt 540 cttcagcaca ggagatgagc acagccgagg gaactggggtcacttggacc aggtggctgc 600 gctgcggtgg gtccaggaca acattgccaa ctttggaggggacccaggct ctgtgaccat 660 ctttggagag tcagcaggag gtcaaagtgt ctctatccttctattatccc ccctgaccaa 720 gaatctcttc catcgagcaa tttccgagag tggcgtggccctcctttcca gtctcttcag 780 gaagaacacc aagtccttgg ctgagaaaat tgccatcgaagctgggtgta aaaccaccac 840 ctcggctgtc atggttcact gcctgcgcca gaagacagaggaagaactca tggaggtgac 900 attgaaaatg aaatttatgg ctctagatct agttggcgaccccaaagaga acaccgcctt 960 cctgaccact gtgattgatg gggtgctgct gccaaaagcacctgcagaga ttctggcaga 1020 gaagaaatac aacatgctgc cctacatggt gggaatcaaccagcaagagt ttggctggat 1080 tatcccaatg caaatgctgg gctatccact ctctgaaggcaaactggacc agaagacagc 1140 tacagaactc ttgtggaagt cctaccccat tgtcaatgtctctaaggagc tgactccagt 1200 ggccactgag aagtatttag gagggacaga tgaccctgtcaaaaagaaag acttgttcct 1260 ggacatgctt gcagatttgt tatttggtgt cccatctgtgaatgtggctc gtcaccacag 1320 agatgctgga gcccccacct atatgtatga gtatcggtatcgcccaagct tctcatcaga 1380 catgagaccc aagacagtga taggggacca tggagatgagatcttctctg tcttaggagc 1440 cccgttttta aaagagggtg ccacagaaga ggagatcaaactgagcaaga tggtgatgaa 1500 atactgggcc aactttgcta ggaatgggaa tcccaatggagaagggcttc ctcaatggcc 1560 agcatatgac tacaaggaag gttacctgca gattggagccaccacccagg cagcccagaa 1620 actgaaagac aaggaagtgg ctttctggac tgagctctgggccaaggagg cagcaaggcc 1680 acgtgagaca gagcacattg agctgtgaat tgaattc 1717<210> SEQ ID NO 21 <211> LENGTH: 565 <212> TYPE: PRT <213> ORGANISM:Oryctolagus cuniculus <400> SEQUENCE: 21 Met Trp Leu Cys Ala Leu Ala LeuAla Ser Leu Ala Ala Cys Thr Ala 1 5 10 15 Trp Gly His Pro Ser Ala ProPro Val Val Asp Thr Val His Gly Lys 20 25 30 Val Leu Gly Lys Phe Val SerLeu Glu Gly Phe Ala Gln Pro Val Ala 35 40 45 Val Phe Leu Gly Val Pro PheAla Lys Pro Pro Leu Gly Ser Leu Arg 50 55 60 Phe Ala Pro Pro Gln Pro AlaGlu Ser Trp Ser His Val Lys Asn Thr 65 70 75 80 Thr Ser Tyr Pro Pro MetCys Ser Gln Asp Ala Val Ser Gly His Met 85 90 95 Leu Ser Glu Leu Phe ThrAsn Arg Lys Glu Asn Ile Pro Leu Lys Phe 100 105 110 Ser Glu Asp Cys LeuTyr Leu Asn Ile Tyr Thr Pro Ala Asp Leu Thr 115 120 125 Lys Arg Gly ArgLeu Pro Val Met Val Trp Ile His Gly Gly Gly Leu 130 135 140 Met Val GlyGly Ala Ser Thr Tyr Asp Gly Leu Ala Leu Ser Ala His 145 150 155 160 GluAsn Val Val Val Val Thr Ile Gln Tyr Arg Leu Gly Ile Trp Gly 165 170 175Phe Phe Ser Thr Gly Asp Glu His Ser Arg Gly Asn Trp Gly His Leu 180 185190 Asp Gln Val Ala Ala Leu Arg Trp Val Gln Asp Asn Ile Ala Asn Phe 195200 205 Gly Gly Asp Pro Gly Ser Val Thr Ile Phe Gly Glu Ser Ala Gly Gly210 215 220 Gln Ser Val Ser Ile Leu Leu Leu Ser Pro Leu Thr Lys Asn LeuPhe 225 230 235 240 His Arg Ala Ile Ser Glu Ser Gly Val Ala Leu Leu SerSer Leu Phe 245 250 255 Arg Lys Asn Thr Lys Ser Leu Ala Glu Lys Ile AlaIle Glu Ala Gly 260 265 270 Cys Lys Thr Thr Thr Ser Ala Val Met Val HisCys Leu Arg Gln Lys 275 280 285 Thr Glu Glu Glu Leu Met Glu Val Thr LeuLys Met Lys Phe Met Ala 290 295 300 Leu Asp Leu Val Gly Asp Pro Lys GluAsn Thr Ala Phe Leu Thr Thr 305 310 315 320 Val Ile Asp Gly Val Leu LeuPro Lys Ala Pro Ala Glu Ile Leu Ala 325 330 335 Glu Lys Lys Tyr Asn MetLeu Pro Tyr Met Val Gly Ile Asn Gln Gln 340 345 350 Glu Phe Gly Trp IleIle Pro Met Gln Met Leu Gly Tyr Pro Leu Ser 355 360 365 Glu Gly Lys LeuAsp Gln Lys Thr Ala Thr Glu Leu Leu Trp Lys Ser 370 375 380 Tyr Pro IleVal Asn Val Ser Lys Glu Leu Thr Pro Val Ala Thr Glu 385 390 395 400 LysTyr Leu Gly Gly Thr Asp Asp Pro Val Lys Lys Lys Asp Leu Phe 405 410 415Leu Asp Met Leu Ala Asp Leu Leu Phe Gly Val Pro Ser Val Asn Val 420 425430 Ala Arg His His Arg Asp Ala Gly Ala Pro Thr Tyr Met Tyr Glu Tyr 435440 445 Arg Tyr Arg Pro Ser Phe Ser Ser Asp Met Arg Pro Lys Thr Val Ile450 455 460 Gly Asp His Gly Asp Glu Ile Phe Ser Val Leu Gly Ala Pro PheLeu 465 470 475 480 Lys Glu Gly Ala Thr Glu Glu Glu Ile Lys Leu Ser LysMet Val Met 485 490 495 Lys Tyr Trp Ala Asn Phe Ala Arg Asn Gly Asn ProAsn Gly Glu Gly 500 505 510 Leu Pro Gln Trp Pro Ala Tyr Asp Tyr Lys GluGly Tyr Leu Gln Ile 515 520 525 Gly Ala Thr Thr Gln Ala Ala Gln Lys LeuLys Asp Lys Glu Val Ala 530 535 540 Phe Trp Thr Glu Leu Trp Ala Lys GluAla Ala Arg Pro Arg Glu Thr 545 550 555 560 Glu His Ile Glu Leu 565<210> SEQ ID NO 22 <211> LENGTH: 6 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Synthetic <400> SEQUENCE: 22 cacgtg 6 <210> SEQID NO 23 <211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Synthetic <400> SEQUENCE: 23 ggcaggaatt ctgccatgtggctctg 26 <210> SEQ ID NO 24 <211> LENGTH: 27 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthetic <400> SEQUENCE: 24cgggaattca cattcacagc tcaatgt 27 <210> SEQ ID NO 25 <211> LENGTH: 6<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: Synthetic <400>SEQUENCE: 25 cacctg 6 <210> SEQ ID NO 26 <211> LENGTH: 543 <212> TYPE:PRT <213> ORGANISM: Oryctolagus cuniculus <400> SEQUENCE: 26 Met Trp LeuCys Ala Leu Ala Leu Ala Ser Leu Ala Ala Cys Thr Ala 1 5 10 15 Trp GlyHis Pro Ser Ala Pro Pro Val Val Asp Thr Val His Gly Lys 20 25 30 Val LeuGly Lys Phe Val Ser Leu Glu Gly Phe Ala Gln Pro Val Ala 35 40 45 Val PheLeu Gly Val Pro Phe Ala Lys Pro Pro Leu Gly Ser Leu Arg 50 55 60 Phe AlaPro Pro Gln Pro Ala Glu Ser Trp Ser His Val Lys Asn Thr 65 70 75 80 ThrSer Tyr Pro Pro Met Cys Ser Gln Asp Ala Val Ser Gly His Met 85 90 95 LeuSer Glu Leu Phe Thr Asn Arg Lys Glu Asn Ile Pro Leu Lys Phe 100 105 110Ser Glu Asp Cys Leu Tyr Leu Asn Ile Tyr Thr Pro Ala Asp Leu Thr 115 120125 Lys Arg Gly Arg Leu Pro Val Met Val Trp Ile His Gly Gly Gly Leu 130135 140 Met Val Gly Gly Ala Ser Thr Tyr Asp Gly Leu Ala Leu Ser Ala His145 150 155 160 Glu Asn Val Val Val Val Thr Ile Gln Tyr Arg Leu Gly IleTrp Gly 165 170 175 Phe Phe Ser Thr Gly Asp Glu His Ser Arg Gly Asn TrpGly His Leu 180 185 190 Asp Gln Val Ala Ala Leu Arg Trp Val Gln Asp AsnIle Ala Asn Phe 195 200 205 Gly Gly Asp Pro Gly Ser Val Thr Ile Phe GlyGlu Ser Ala Gly Gly 210 215 220 Gln Ser Val Ser Ile Leu Leu Leu Ser ProLeu Thr Lys Asn Leu Phe 225 230 235 240 His Arg Ala Ile Ser Glu Ser GlyVal Ala Leu Leu Ser Ser Leu Phe 245 250 255 Arg Lys Asn Thr Lys Ser LeuAla Glu Lys Ile Ala Ile Glu Ala Gly 260 265 270 Cys Lys Thr Thr Thr SerAla Val Met Val His Cys Leu Arg Gln Lys 275 280 285 Thr Glu Glu Glu LeuMet Glu Val Thr Leu Lys Met Lys Phe Met Ala 290 295 300 Leu Asp Leu ValGly Asp Pro Lys Glu Asn Thr Ala Phe Leu Thr Thr 305 310 315 320 Val IleAsp Gly Val Leu Leu Pro Lys Ala Pro Ala Glu Ile Leu Ala 325 330 335 GluLys Lys Tyr Asn Met Leu Pro Tyr Met Val Gly Ile Asn Gln Gln 340 345 350Glu Phe Gly Trp Ile Ile Pro Met Gln Met Leu Gly Tyr Pro Leu Ser 355 360365 Glu Gly Lys Leu Asp Gln Lys Thr Ala Thr Glu Leu Leu Trp Lys Ser 370375 380 Tyr Pro Ile Val Asn Val Ser Lys Glu Leu Thr Pro Val Ala Thr Glu385 390 395 400 Lys Tyr Leu Gly Gly Thr Asp Asp Pro Val Lys Lys Lys AspLeu Phe 405 410 415 Leu Asp Met Leu Ala Asp Leu Leu Phe Gly Val Pro SerVal Asn Val 420 425 430 Ala Arg His His Arg Asp Ala Gly Ala Pro Thr TyrMet Tyr Glu Tyr 435 440 445 Arg Tyr Arg Pro Ser Phe Ser Ser Asp Met ArgPro Lys Thr Val Ile 450 455 460 Gly Asp His Gly Asp Glu Ile Phe Ser ValLeu Gly Ala Pro Phe Leu 465 470 475 480 Lys Glu Gly Ala Thr Glu Glu GluIle Lys Leu Ser Lys Met Val Met 485 490 495 Lys Tyr Trp Ala Asn Phe AlaArg Asn Gly Asn Pro Asn Gly Glu Gly 500 505 510 Leu Pro Gln Trp Pro AlaTyr Asp Tyr Lys Glu Gly Tyr Leu Gln Ile 515 520 525 Gly Ala Thr Thr GlnAla Ala Gln Lys Leu Lys Asp Lys Glu Val 530 535 540 <210> SEQ ID NO 27<211> LENGTH: 2191 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400>SEQUENCE: 27 tcgagcggcc acccgggcag gtctctgggt gaatagcagc gtgtccgccggcagcgaacc 60 gagaccagcg agccgaccat gcggctgcac agacttcgtg cgcggctgagcgcggtggcc 120 tgtgggcttc tgctgcttct tgtccggggc cagggccagg actcagccagtcccatccgg 180 accacacaca cggggcaggt gctggggagt cttgtccatg tgaagggcgccaatgccggg 240 gtccaaacct tcctgggaat tccatttgcc aagccacctc taggtccgctgcgatttgca 300 ccccctgagc cccctgaatc ttggagtggt gtgagggatg gaaccacccatccggccatg 360 tgtctacagg acctcaccgc agtggagtca gagtttctta gccagttcaacatgaccttc 420 ccttccgact ccatgtctga ggactgcctg tacctcagca tctacacgccggcccatagc 480 catgaaggct ctaacctgcc ggtgatggtg tggatccacg gtggtgcgcttgtttttggc 540 atggcttcct tgtatgatgg ttccatgctg gctgccttgg agaacgtggtggtggtcatc 600 atccagtacc gcctgggtgt cctgggcttc ttcagcactg gagacaagcacgcaaccggc 660 aactggggct acctggacca agtggctgca ctacgctggg tccagcagaatatcgcccac 720 tttggaggca accctgaccg tgtcaccatt tttggcgagt ctgcgggtggcacgagtgtg 780 tcttcgcttg ttgtgtcccc catatcccaa ggactcttcc acggagccatcatggagagt 840 ggcgtggccc tcctgcccgg cctcattgcc agctcagctg atgtcatctccacggtggtg 900 gccaacctgt ctgcctgtga ccaagttgac tctgaggccc tggtgggctgcctgcggggc 960 aagagtaaag aggagattct tgcaattaac aagcctttca agatgatccccggagtggtg 1020 gatggggtct tcctgcccag gcacccccag gagctgctgg cctctgccgactttcagcct 1080 gtccctagca ttgttggtgt caacaacaat gaattcggct ggctcatccccaaggtcatg 1140 aggatctatg atacccagaa ggaaatggac agagaggcct cccaggctgctctgcagaaa 1200 atgttaacgc tgctgatgtt gcctcctaca tttggtgacc tgctgagggaggagtacatt 1260 ggggacaatg gggatcccca gaccctccaa gcgcagttcc aggagatgatggcggactcc 1320 atgtttgtga tccctgcact ccaagtagca cattttcagt gttcccgggcccctgtgtac 1380 ttctacgagt tccagcatca gcccagctgg ctcaagaaca tcaggccaccgcacatgaag 1440 gcagaccatg gtgatgagct tccttttgtt ttcagaagtt tctttgggggcaactacatt 1500 aaattcactg aggaagagga gcagctaagc aggaagatga tgaagtactgggccaacttt 1560 gcgagaaatg ggaaccccaa tggcgagggt ctgccacact ggccgctgttcgaccaggag 1620 gagcaatacc tgcagctgaa cctacagcct gcggtgggcc gggctctgaaggcccacagg 1680 ctccagttct ggaagaaggc gctgccccaa aagatccagg agctcgaggagcctgaagag 1740 agacacacag agctgtagct ccctgtgccg gggaggaggg ggtgggttcgctgacaggcg 1800 agggtcagcc tgctgtgccc acacacaccc actaaggaga aagaagttgattccttcatt 1860 cacttcgcca ttcattcata cttccgtcca gaagttgatt ccttcattcacttcgccatt 1920 cattcatact tccgtccatc cattcagaaa ccggyattta ttaagaatttactcaggcat 1980 gatggcccat acttgtaatc ccagctattg ggaaggatga gatgggaggatggcttgagg 2040 ccagaggttt gagaccgacc agccagggca acacagtgag accccttctcaaaaaaaaaa 2100 aaaaaaaaag agagagtgtg tgattagaag ctaaatagga aagttttgagcttcaagtca 2160 gtgaggagta aaaaagattt ttaaaaagca a 2191 <210> SEQ ID NO28 <211> LENGTH: 559 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400>SEQUENCE: 28 Met Arg Leu His Arg Leu Arg Ala Arg Leu Ser Ala Val Ala CysGly 1 5 10 15 Leu Leu Leu Leu Leu Val Arg Gly Gln Gly Gln Asp Ser AlaSer Pro 20 25 30 Ile Arg Thr Thr His Thr Gly Gln Val Leu Gly Ser Leu ValHis Val 35 40 45 Lys Gly Ala Asn Ala Gly Val Gln Thr Phe Leu Gly Ile ProPhe Ala 50 55 60 Lys Pro Pro Leu Gly Pro Leu Arg Phe Ala Pro Pro Glu ProPro Glu 65 70 75 80 Ser Trp Ser Gly Val Arg Asp Gly Thr Thr His Pro AlaMet Cys Leu 85 90 95 Gln Asp Leu Thr Ala Val Glu Ser Glu Phe Leu Ser GlnPhe Asn Met 100 105 110 Thr Phe Pro Ser Asp Ser Met Ser Glu Asp Cys LeuTyr Leu Ser Ile 115 120 125 Tyr Thr Pro Ala His Ser His Glu Gly Ser AsnLeu Pro Val Met Val 130 135 140 Trp Ile His Gly Gly Ala Leu Val Phe GlyMet Ala Ser Leu Tyr Asp 145 150 155 160 Gly Ser Met Leu Ala Ala Leu GluAsn Val Val Val Val Ile Ile Gln 165 170 175 Tyr Arg Leu Gly Val Leu GlyPhe Phe Ser Thr Gly Asp Lys His Ala 180 185 190 Thr Gly Asn Trp Gly TyrLeu Asp Gln Val Ala Ala Leu Arg Trp Val 195 200 205 Gln Gln Asn Ile AlaHis Phe Gly Gly Asn Pro Asp Arg Val Thr Ile 210 215 220 Phe Gly Glu SerAla Gly Gly Thr Ser Val Ser Ser Leu Val Val Ser 225 230 235 240 Pro IleSer Gln Gly Leu Phe His Gly Ala Ile Met Glu Ser Gly Val 245 250 255 AlaLeu Leu Pro Gly Leu Ile Ala Ser Ser Ala Asp Val Ile Ser Thr 260 265 270Val Val Ala Asn Leu Ser Ala Cys Asp Gln Val Asp Ser Glu Ala Leu 275 280285 Val Gly Cys Leu Arg Gly Lys Ser Lys Glu Glu Ile Leu Ala Ile Asn 290295 300 Lys Pro Phe Lys Met Ile Pro Gly Val Val Asp Gly Val Phe Leu Pro305 310 315 320 Arg His Pro Gln Glu Leu Leu Ala Ser Ala Asp Phe Gln ProVal Pro 325 330 335 Ser Ile Val Gly Val Asn Asn Asn Glu Phe Gly Trp LeuIle Pro Lys 340 345 350 Val Met Arg Ile Tyr Asp Thr Gln Lys Glu Met AspArg Glu Ala Ser 355 360 365 Gln Ala Ala Leu Gln Lys Met Leu Thr Leu LeuMet Leu Pro Pro Thr 370 375 380 Phe Gly Asp Leu Leu Arg Glu Glu Tyr IleGly Asp Asn Gly Asp Pro 385 390 395 400 Gln Thr Leu Gln Ala Gln Phe GlnGlu Met Met Ala Asp Ser Met Phe 405 410 415 Val Ile Pro Ala Leu Gln ValAla His Phe Gln Cys Ser Arg Ala Pro 420 425 430 Val Tyr Phe Tyr Glu PheGln His Gln Pro Ser Trp Leu Lys Asn Ile 435 440 445 Arg Pro Pro His MetLys Ala Asp His Gly Asp Glu Leu Pro Phe Val 450 455 460 Phe Arg Ser PhePhe Gly Gly Asn Tyr Ile Lys Phe Thr Glu Glu Glu 465 470 475 480 Glu GlnLeu Ser Arg Lys Met Met Lys Tyr Trp Ala Asn Phe Ala Arg 485 490 495 AsnGly Asn Pro Asn Gly Glu Gly Leu Pro His Trp Pro Leu Phe Asp 500 505 510Gln Glu Glu Gln Tyr Leu Gln Leu Asn Leu Gln Pro Ala Val Gly Arg 515 520525 Ala Leu Lys Ala His Arg Leu Gln Phe Trp Lys Lys Ala Leu Pro Gln 530535 540 Lys Ile Gln Glu Leu Glu Glu Pro Glu Glu Arg His Thr Glu Leu 545550 555 <210> SEQ ID NO 29 <211> LENGTH: 31 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthetic <400> SEQUENCE: 29cggtctagag agctacagct ctgtgtgtct g 31 <210> SEQ ID NO 30 <211> LENGTH:31 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic<400> SEQUENCE: 30 cgagtctaga gagccgacca tgcggctgca c 31

What is claimed is:
 1. A method for sensitizing tumor cells to achemotherapeutic prodrug APC or CPT-11 in vitro comprising transfectingselected tumor cells with a composition comprising an isolatedpolynucleotide encoding a carboxylesterase wherein said carboxylesteraseis operably linked to a promoter that directs expression of saidcarboxylesterase in said tumor cells, and wherein expression of thecarboxylesterase renders the tumor cells more sensitive to the cytotoxiceffect of said chemotherapeutic prodrug APC or CPT-11.
 2. A method ofinhibiting tumor cell growth in vitro comprising: (A) sensitizing tumorcells in accordance with the method of claim 1; and (B) contacting saidsensitized tumor cells with said chemotherapeutic prodrug CPT-11 or APCso that tumor cell growth is inhibited.
 3. The method according to claim1 wherein the carboxylesterase is selected from the group consisting ofrabbit carboxylesterase and human intestinal carboxylesterase.
 4. Themethod according to claim 1 wherein the carboxylesterase is delivered byan adenoviral vector.